坛紫菜碳酸酐酶基因的克隆、表达及酶活性分析  

Gene cloning, expression and enzyme activity analysis of the carbonic anhydrase from Porphyra haitanensis(Rhodophyta)

作　　者：郇丽[1,2] 贾兆君[3] 张宝玉[1] 牛建峰[1] 林阿朋[1] 何林文[1] 王广策[1] 

机构地区：[1]中国科学院海洋研究所,山东青岛266071 [2]中国科学院大学研究生院,北京100049 [3]大连理工大学生命科学与技术学院,辽宁大连116024

出　　处：《海洋科学》2014年第8期8-15,共8页Marine Sciences

基　　金：公益性行业(农业)科研专项(200903030);国家自然科学基金(41176134);海洋公益性行业科研专项项目(201105023-7);国家973计划前期专项(2011CB411908)

摘　　要：以坛紫菜丝状体为材料,采用RACE方法获得坛紫菜碳酸酐酶(CA)基因的全长cDNA。该cDNA全长1 081 bp,具有一个825 bp的开放阅读框,可编码274个氨基酸。序列同源性分析显示该cDNA序列推导的氨基酸序列与其他物种的碳酸酐酶具有较高的一致性,其中与条斑紫菜的一致性达到96%。氨基酸序列分析表明该蛋白为β-CA,含有两个CA活性位点,无跨膜结构,可能存在一个信号肽将其定位到叶绿体中,与藻类和细菌聚类。原核诱导表达得到一个72 kDa左右的融合蛋白,酶活测定结果显示此蛋白具有碳酸酐酶活性。该实验对进一步深入研究坛紫菜CA的功能及坛紫菜碳代谢、光合作用等生理过程具有重要的参考价值。Carbonic anhydrase （CA）, a zinc-containing enzyme is widespread in living organisms, catalyses the reversible hydration of CO2 and 3HCO-. In this study, a full-length cDNA of CA was isolated from Porphyra haitanensis with rapid amplification of cDNA ends （RACE）. This sequence was 1 081 bp in length and encodes a polypeptide of 274 amino acid residues. The deduced polypeptide showed high identities with the CA genes ranging from unicellular algae and bacteria to green plant. Phylogenetic tree analysis showed that the CA gene from P. haitanensis was more closely assembled with algae and bacteria. A ~72 kDa fused protein was obtained by the recombinant prokaryotic expression and the enzyme activity analysis showed that it had the activity of CA.

关 键 词：坛紫菜 碳酸酐酶 序列分析 原核表达 酶活性 

分 类 号：Q78[生物学—分子生物学]